Important new actions of erythropoietin (EPO) are increasingly being realized in the contexts of ischemic cytoprotection, angiogenesis, thrombosis and the anemia of cancer and chemotherapy. To understand how EPO exerts these effects; to identify potentially new candidate targets for anti-anemia agents (especially in cancer contexts); and to discern between beneficial vs potentially adverse properties of EPO and emerging ESAs, an improved understanding of EPO receptor (EPOR) action mechanisms is needed. Towards revealing new EPO effects, the PI has applied a unique combination of primary erythroid progenitor cell development systems, knocked-in EPO receptor alleles, and global transcriptome profiling approaches. These aggressive investigations have led to the discovery of several intriguing new EPO/EPOR response circuits, and action modes. This includes sharp EPO/ EPOR modulation of novel cell surface adhesion / migration factors, cell cycle regulators, and survival factors. This R01-A1 renewal application builds on these discoveries, and major new progress. Aim 1 will extend transcriptome- based analyses to define novel EPOR circuits in the contexts of progenitor cell stage specific- responses, and EPOR- PY343 / STAT5- directed pathways. Aims 2- 4 then focus on three of our newly discovered EPO/EPOR response factors which regulate adhesion / migration; erythroid progenitor expansion; and cell death. Aim 2 specifically will employ unique knock-in and knock-out models plus stromal cell interaction assays to assess regulation of pro-erythroblast expansion and/or migration by PODOCALYXIN (PODXL), a CD34-related sialomucin and prime new EPO response gene. Aim 3 will apply a novel knock-out model to discover how Serpina-3G (S3G) acts as a strongly EPO-induced intracellular serpin (and predicted Ser/Cys protease inhibitor) to selectively support erythropoiesis during anemia. Via co-IP and mass spectrometry, S3G's target protease also will be defined. Aim 4 will use new knock-out and transgenic models to define death activating mechanisms that the EPO-regulated death- associated protein kinase, DAPK2, engages to limit EPOR- dependent erythropoiesis. Notably, PODXL, Serpina-3G and DAPK2 each are regulated via an EPOR PY343 / STAT5 axis which now has been shown by three laboratories (including, first, the PI's) to be critical for erythropoiesis during anemia and /or embryogenesis. To further underscore progress, the PI has also now published four additional new papers in BLOOD (since the submission of this application in 2007) in direct support of each proposed aim. Proposed investigations should markedly advance a molecular understanding of EPO/EPOR- dependent erythropoiesis, and provide important new information, resources, reagents and models for present and future studies of key erythropoietic regulators. PUBLIC HEALTH RELEVANCE: Erythropoietin (EPO) is used with excellent success to treat the anemia of chronic renal disease, and basic aspects of EPO's action mechanisms are well-studied. Recently, strong evidence has been provided for new roles for EPO in cytoprotection against ischemic damage, and in the compromising contexts of cancer progression, and thrombolytic events. To understand how EPO (and emerging EPO mimetics) exert such effects, a better understanding of EPO response pathways is required: Proposed investigations respond to this need, and will apply aggressive gene profiling approaches and novel mouse models to define new EPO/EPOR response pathways in primary erythroid progenitor cell systems.